1. Field of the Invention
The invention is directed to purified and isolated novel murine and human kinase polypeptides and fragments thereof, the nucleic acids encoding such polypeptides, processes for production of recombinant forms of such polypeptides, fragmented peptides derived from these polypeptides, antibodies generated against these polypeptides, methods of identifying activators and inhibitors of the activity of these kinases, and therapeutic and diagnostic uses thereof.
2. Background
Cells respond to external signals and internal signals, such as those produced by disease conditions, by activating cellular signaling pathways. Cellular signaling often involves a molecular activation cascade, during which a receptor propagates a ligand-receptor mediated signal by specifically activating intracellular protein kinases which phosphorylate target substrates. These substrates can themselves be kinases which become activated following phosphorylation.
The eukaryotic protein kinases make up a large and rapidly expanding family of proteins related on the basis of homologous catalytic domains. Spurred by the development of gene cloning and sequencing methodologies, distinct protein kinase genes have been identified from a wide selection of invertebrates and lower eukaryotes, including Drosophila, Caenorhabditis elegans, Aplysia, Hydra, Dictyostelium, and budding (Saccharomyces cerevisiae) and fission (Schizosaccharomyces pombe) yeast. Homologous genes have also been identified in higher plants. Protein kinases, however, are not limited to the eukaryotes. Enzyme activities have been well documented in prokaryotes, but the prokaryotic protein kinase genes are not obviously similar to those of the eukaryotes.
Given the important function of kinases in general, there is a need in the art for additional members of the kinase family. In addition, in view of the continuing interest in protein research, the discovery, identification, and roles of new proteins, such as protein kinases, are at the forefront of modem molecular biology and biochemistry. Despite the growing body of knowledge, there is still a need in the art for the identity and function of proteins having kinase activities. In addition, because there is an unmet need for therapeutic compounds which modulate kinase activity and because protein kinases are useful biochemical reagents, there is also need in the art for the continued discovery of unique members of the protein kinase family and potential therapeutic targets thereof.
The invention aids in fulfilling these various needs in the art by providing isolated murine and human kinase nucleic acids and polypeptides encoded by these nucleic acids. Particular embodiments of the invention are directed to isolated murine and human kinase nucleic acid molecules comprising the DNA sequences of SEQ ID NOs:1-7 and isolated murine and human kinase nucleic acid molecules encoding the amino acid sequences of SEQ ID NOs:8-14, as well as nucleic acid molecules complementary to these sequences. Both single-stranded and double-stranded RNA and DNA nucleic acid molecules are encompassed by the invention, as well as nucleic acid molecules that hybridize to a denatured, double-stranded DNA comprising all or a portion of SEQ ID NOs:1-7. Also encompassed are isolated nucleic acid molecules that are derived by in vitro mutagenesis of nucleic acid molecules comprising sequences of SEQ ID NOs:1-7, that are degenerate from nucleic acid molecules comprising sequences of SEQ ID NOs:1-7, and that are allelic variants of DNA of the invention. The invention also encompasses recombinant vectors that direct the expression of these nucleic acid molecules and host cells stably or transiently transformed or transfected with these vectors.
In addition, the invention encompasses methods of using the nucleic acids noted above to identify nucleic acids encoding proteins having kinase activity and to study cell signal transduction.
The invention also encompasses the use of sense or antisense oligonucleotides from the nucleic acid of SEQ ID NOs:1-7 to inhibit the expression of the polynucleotide encoded by the kinase genes.
The invention also encompasses isolated polypeptides and fragments thereof encoded by these nucleic acid molecules including soluble polypeptide portions of SEQ ID NOs:8-14. The invention further encompasses methods for the production of these polypeptides, including culturing a host cell under conditions promoting expression and recovering the polypeptide from the culture medium. Especially, the expression of these polypeptides in bacteria, yeast, plant, insect, and animal cells is encompassed by the invention.
In general, the polypeptides of the invention can be used to study cellular processes such as signal transduction and to screen for compounds which modulate kinase activity which may have therapeutic potential. In addition, these polypeptides can be used to identify proteins associated with the polypeptides of the invention.
In addition, the invention includes assays utilizing these polypeptides to screen for potential inhibitors of activity associated with polypeptide counter-structure molecules, and methods of using these inhibitors as therapeutic agents for the treatment of cancer and other proliferative diseases and diseases mediated by polypeptide counter-structure molecules. Further, methods of using these polypeptides in the design of inhibitors thereof are also an aspect of the invention.
The invention further provides a method for using these polypeptides as molecular weight markers that allow the estimation of the molecular weight of a protein or a fragmented protein, as well as a method for the visualization of the molecular weight markers of the invention thereof using electrophoresis. The invention further encompasses methods for using the polypeptides of the invention as markers for determining the isoelectric point of an unknown protein, as well as controls for establishing the extent of fragmentation of a protein.
Further encompassed by this invention are kits to aid in these determinations.
Further encompassed by this invention is the use of the kinase nucleic acid sequences, predicted amino acid sequences of the polypeptide or fragments thereof, or a combination of the predicted amino acid sequences of the polypeptide and fragments thereof for use in searching an electronic database to aid in the identification of sample nucleic acids and/or proteins.
Isolated polyclonal or monoclonal antibodies that bind to these polypeptides are also encompassed by the invention, in addition the use of these antibodies to aid in purifying the murine and human kinase polypeptides.
Also encompassed by the invention is a method of designing an inhibitor of the kinase polypeptide of the invention, the method comprising the steps of determining the three-dimensional structure of such polypeptide, analyzing the three-dimensional structure for the likely binding sites of substrates, synthesizing a molecule that incorporates a predicted reactive site, and determining the polypeptide-inhibiting activity of the molecule.
Another aspect of the invention is a method for identifying compounds that activate or inhibit kinase activity comprising:
(a) bringing a test compound into contact with the polypeptide of the invention and a substrate; and
(b) determining whether the test compound activates or inhibits the kinase activity of said polypeptide. In a preferred embodiment of this method, the test compound is brought into contact with the polypeptide in a cell containing at least one recombinant vector that directs the expression of at least one polynucleotide encoding said polypeptide.
Another aspect of the invention is a method for inhibiting the kinase activity of the polypeptide of the invention comprising forming a mixture of the polypeptide, a substrate, and a compound, wherein the compound blocks the binding of the polypeptide with the substrate.